A conventional DNA sequencing gel matrix typically contains 3-9 M urea, or a combination of urea and formamide as a denaturant. The function of a denaturant in a gel is to help keep DNA molecules denatured during electrophoresis in order to achieve accurate base calling. The existence of urea or formamide in a gel matrix represents a distinctive difference between denaturing gel electrophoresis for DNA sequencing (separating single-stranded DNA) and non-denaturing gel electrophoresis for separating double stranded DNA. The denaturing power of a gel matrix is generally proportional to the concentration of a denaturant in the gel matrix. Higher denaturing powers minimize compression, a self-folding behavior, of single stranded DNA fragments in a DNA sequencing sample.
When urea is used, however, the denaturing power is limited by the saturation concentration of urea, which is about 9M. When formamide is used, there is also a limit, which is the manageable viscosity of a matrix and separation speed. For example, because the viscosity of the matrix increases significantly with the percentage of formamide in a gel, separation speed decreases with higher percentages of formamide. Sometimes, the denaturing power of a gel with a maximum concentration of urea or formamide still does not provide sufficient denaturing power to resolve some compression bands in GC-rich DNA samples.
A popular method to overcome the above-mentioned problem of insufficient denaturing power is to heat up a gel during electrophoresis, typically 35-70° C., and add a denaturant. The combination of high temperature electrophoresis and high concentration denaturant typically provides sufficient denaturing power to resolve difficult compression bands.
There are several issues, however, associated with electrophoresis using a matrix containing urea or formamide. First, urea and formamide degrade in the basic solution that is typically used for DNA sequencing (pH 8.0-8.5). Higher temperatures accelerate such degradation. The degradation of urea or formamide has adverse effects on the gel and separation columns. The degradation products include ammonia, uric acid, and formic acid. These products increase the ion concentration and pH of the matrix. They may also cause bubble formation in a matrix at higher temperatures. When an uncoated capillary is used, these degradation products reduce the adhering affinity between the polymer dynamic coating and the capillary wall. This allows electroosmotic flow to occur, which consequently reduces separation efficiency. Capillary lifetime is also shortened because the decreased coverage of polymer coating on the capillary wall allows biomolecules to attack and adsorb onto the capillary wall, which in turn degrades separation efficiency.
To minimize these problems, one can take several approaches: a) optimize the electric field strength and column temperature so that the degradation products can be consistently driven out of the separation column at a rate that is equivalent to or higher than the rate of generation; b) develop better dynamic coating polymers that adsorb onto capillary wall more efficiently under high temperature; c) reduce the pH value of the gel matrix, e.g., from pH 8.3 down to pH 7.6,in order to reduce the degradation rate of urea or formamide at high temperatures, and to enhance the adsorbing efficiency of polymer on the capillary wall. These methods, however, have limitations.